Neurodegeneration is closely linked to perturbations in protein quality control. Ubiquitin pathways are central to protein quality control through their role in protein degradation. Here, I will investigate how deubiquitinating enzymes (DUBs), integral components of ubiquitin-dependent pathways, are involved in neurodegenerative processes and how DUBs themselves are regulated in turn. These studies focus on a unique DUB and the polyglutamine disease protein in Spinocerebellar Ataxia Type 3, ataxin-3 (ATS), and USP25 and USP28, two other brain-expressed DUBs that share domains with ATS. ATS is a DUB that functions in protein quality control. I previously established that endogenous ATS is ubiquitinated and its ubiquitination increases in proteotoxic stress. Importantly, ubiquitination directly enhances ATS's catalytic activity. During the K99 phase I determined where ATS is ubiquitinated, gathered nsight into the mechanism behind its ubiquitination-dependent activation, and collected evidence that ATS ubiquitination is important for some of its cellular functions. Since ATS is neuroprotective in fly models of neurodegeneration, during the ROO phase I will use Drosophila melanogaster models of neurodegeneration to explore the role of ATS ubiquitination in its cytoprotective functions. I will generate fly lines expressing wild type or non-ubiquitinatable ATS throughout the nervous system or in the eye. Through structural, behavioral and electrophysiological studies I will examine whether ATS ubiquitination is important for its neuroprotective role in intact animals. Complementary studies in mammalian cell culture will investigate the importance of ATS ubiquitination in its ability to protect cells during proteotoxic stress. USP25 and USP28 are two DUBs that, like ATS, have ubiquitin-interacting motifs (UIMs). UIMs facilitate host protein ubiquitination; in fact, USP25 is ubiquitinated in cells. My preliminary data indicate that USP25 and USP28 regulate cellular levels of Alzheimer's Disease-causing, mutant Amyloid Precursor Protein (APP). Here, I will investigate the mechanisms by which USP25 and USP28 regulate APP by using cell-based and in vitro assays. I will also examine how USP25 ubiquitination is involved in this cellular function. Finally, I will begin to establish Drosophila assays to examine the role of other DUBs in neurodegeneration in the future.